Signal-mediated Pathway Research Articles

Direct Reprogramming of Mouse Fibroblasts to Osteoblast-like Cells Using Runx2/Dlx5 Factors on Engineered Stiff Hydrogels.

Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles.

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer

Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich’s ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed “PuRepeatFinder.pl” algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein–protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.

Interactions of Auxinic Compounds on Ca2+ Signaling and Root Growth in <i>Arabidopsis thaliana</i>

Auxinic-like compounds have been widely used as weed control agents. Over the years, the modes of action of auxinic herbicides have been elucidated, but most studies thus far have focused on their effects on later stages of plant growth. Here, we show that some select auxins and auxiniclike herbicides trigger a rapid elevation in root cytosolic calcium levels within seconds of application. Arabidopsis thaliana plants expressing the Yellow-Cameleon (YC) 3.60 calcium reporter were treated with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthalene acetic acid (NAA), and two synthetic herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and mecoprop [2-(4-chloro- 2-methylphenoxy) propanoic acid], followed by monitoring cytosolic calcium changes over a 10 minute time course. Seconds after application of compounds to roots, the Ca2+ signaling-mediated pathway was triggered, initiating the plant response to these compounds as monitored and recorded using Fluorescence Resonance Energy Transfer (FRET)-sensitized emission imaging. Each compound elicited a specific and unique cytosolic calcium signature. Also primary root development and elongation was greatly reduced or altered when exposed at two concentrations (0.10 and 1.0 μM) of each compound. Within 20 to 25 min after triggering of the Ca2+ signal, root growth inhibition could be detected. We speculate that differences in calcium signature among the tested auxins and auxinic herbicides might correlate with their variation and potency with regard to root growth inhibition.

Xanomeline suppresses excessive pro-inflammatory cytokine responses through neural signal-mediated pathways and improves survival in lethal inflammation.

Inflammatory conditions characterized by excessive immune cell activation and cytokine release, are associated with bidirectional immune system-brain communication, underlying sickness behavior and other physiological responses. The vagus nerve has an important role in this communication by conveying sensory information to the brain, and brain-derived immunoregulatory signals that suppress peripheral cytokine levels and inflammation. Brain muscarinic acetylcholine receptor (mAChR)-mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of centrally-acting mAChR agonists is unexplored. To provide insight we used the centrally-acting M1 mAChR agonist xanomeline, previously developed in the context of Alzheimer's disease and schizophrenia. Intraperitoneal administration of xanomeline significantly suppressed serum and splenic TNF levels, alleviated sickness behavior, and increased survival during lethal murine endotoxemia. The anti-inflammatory effects of xanomeline were brain mAChR-mediated and required intact vagus nerve and splenic nerve signaling. The anti-inflammatory efficacy of xanomeline was retained for at least 20h, associated with alterations in splenic lymphocyte, and dendritic cell proportions, and decreased splenocyte responsiveness to endotoxin. These results highlight an important role of the M1 mAChR in a neural circuitry to spleen in which brain cholinergic activation lowers peripheral pro-inflammatory cytokines to levels favoring survival. The therapeutic efficacy of xanomeline was also manifested by significantly improved survival in preclinical settings of severe sepsis. These findings are of interest for strategizing novel therapeutic approaches in inflammatory diseases.

New Strategies in Neuroprotection and Neurorepair

There are currently few clinical strategies in place, which provide effective neuroprotection and repair, despite an intense international effort over the past decades. One possible explanation for this is that a deeper understanding is required of how endogenous mechanisms act to confer neuroprotection. This mini-review reports the proceedings of a recent workshop "Neuroprotection and Neurorepair: New Strategies" (Iguazu Falls, Misiones, Argentina, April 11-13, 2011, Satellite Symposium of the V Neurotoxicity Society Meeting, 2011) in which four areas of active research were identified to have the potential to generate new insights into this field. Topics discussed were (i) metallothionein and other multipotent neuroprotective molecules; (ii) oxidative stress and their signal mediated pathways in neuroregeneration; (iii) neurotoxins in glial cells, and (iv) drugs of abuse with neuroprotective effects.

Mucin Secretion Induced by Titanium Dioxide Nanoparticles

Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO2), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO2 NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO2 NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO2 NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca2+ signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca2+ concentration were monitored with Rhod-2, a fluorescent Ca2+ dye. We found that TiO2 NP-evoked mucin secretion was a function of increasing intracellular Ca2+ concentration resulting from an extracellular Ca2+ influx via membrane Ca2+ channels and cytosolic ER Ca2+ release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca2+ signal and in sustaining a cytosolic Ca2+ increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.

Methods and assays to investigate nuclear export.

A hallmark of eukaryotic cells is their spatial and functional separation into the nucleus and the cytoplasm by the nuclear envelope. Although this separation introduces a potent and sophisticated level of regulation not existing in prokaryotes, it also requires a highly effective and selective transport machinery. All known transport between the nucleus and the cytoplasm occurs through the nuclear pore complex (NPC) (for reviews, see Fabre and Hurt 1997; Gant et al. 1998; Pante and Aebi 1996). Theoretically, proteins with masses <40kDa can enter and leave the nucleus by passive diffusion. However, even most of the smaller proteins and nucleic acids appear to be transported by signal-mediated pathways, probably because signal-mediated trafficking is more efficient and more amenable to specific regulation than diffusion. The most prevalent nuclear export signals (NESs) found consist of a short leucine-rich stretch of amino acids in which the leucine residues are critical for function (for review, see GÖrlich and Kutay 1999; Mattaj and Englmeier 1998 and references therein). Leucine-rich NESs have been identified in an increasing number of cellular and viral proteins executing quite heterologous biological functions. As extensively reviewed in the individual chapters of this book, viruses efficiently exploit the cellular transport machinery in order to successfully parasitize the cell. This chapter will summarize existing in vitro and in vivo assay systems to study nuclear export in mammalian cells, focusing especially on novel experimental approaches. As a result of space limitation, important primary papers will not be quoted and we apologize to our colleagues for these omissions.KeywordsNuclear ExportNuclear ImportNuclear Pore ComplexNuclear Export SignalNucleocytoplasmic TransportThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Subcellular Localization of the Aryl Hydrocarbon Receptor Is Modulated by the Immunophilin Homolog Hepatitis B Virus X-associated Protein 2

The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serving a largely unknown function. Transiently expressed AhR.YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR.YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that approximately 25% of transiently expressed AhR was associated with XAP2, in contrast with approximately 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that approximately 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophilin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-mediated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of the AhR.

Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.

The HIV-1 Rev protein.

The nuclear export of intron-containing HIV-1 RNA is critically dependent on the activity of Rev, a virally encoded sequence-specific RNA-binding protein. Rev shuttles between the nucleus and the cytoplasm and harbors both a nuclear localization signal and a nuclear export signal. These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export. HIV-1 Rev therefore represents an excellent system with which to study aspects of transport across the nuclear envelope.

Cross-linker dynamics determine the mechanical properties of actin gels

To evaluate the contributions of cross-linker dynamics and polymer deformation to the frequency-dependent stiffness of actin filament gels, we compared the rheological properties of actin gels with three types of cross-linkers: a weak one, Acanthamoeba alpha-actinin (dissociation rate constant 5.2 s-1, association rate constant 1.1 x 10(6) M-1 s-1); a strong one, chicken smooth muscle alpha-actinin (dissociation rate constant 0.66 s-1, association rate constant 1.20 x 10(6) M-1 s-1); and an extremely strong one, biotin/avidin (dissociation rate constant approximately zero). The biotin/avidin cross-linked gel, whose behavior is determined by polymer bending alone, behaves like a solid and shows no frequency dependence. The amoeba alpha-actinin cross-linked gel behaves like a viscoelastic fluid, and the frequency dependence of the stiffness can be explained by a mathematical model for dynamically cross-linked gels. The stiffness of the chicken alpha-actinin cross-linked gel is independent of frequency, and has viscoelastic properties intermediate between the two. The two alpha-actinins have similar association rate constants for binding to actin filaments, consistent with a diffusion-limited reaction. Rigid cross-links make the gel stiff, but make it elastic without the ability to deform permanently. Dynamically cross-linked actin filaments should allow the cell to react passively to various outside forces without any sort of signaling. Slower, signal-mediated pathways, such as severing filaments or changing the affinity of cross-linkers, could alter the nature of these passive reactions.